High throughput screening assay systems in microscale fluidic devices

ABSTRACT

The present invention provides novel microfluidic devices and methods that are useful for performing high-throughput screening assays. In particular, the devices and methods of the invention are useful in screening large numbers of different compounds for their effects on a variety of chemical, and preferably, biochemical systems.

BACKGROUND OF THE INVENTION

[0001] There has long been a need for the ability to rapidly assaycompounds for their effects on various biological processes. Forexample, enzymologists have long sought better substrates, betterinhibitors or better catalysts for enzymatic reactions. Similarly, inthe pharmaceutical industries, attention has been focused on identifyingcompounds that may block, reduce, or even enhance the interactionsbetween biological molecules. Specifically, in biological systems, theinteraction between a receptor and its ligand often may result, eitherdirectly or through some downstream event, in either a deleterious orbeneficial effect on that system, and consequently, on a patient forwhom treatment is sought. Accordingly, researchers have long soughtafter compounds or mixtures of compounds that can reduce, block or evenenhance that interaction.

[0002] Modern drug discovery is limited by the throughput of the assaysthat are used to screen compounds that possess these described effects.In particular, screening of the maximum number of different compoundsnecessitates reducing the time and labor requirements associated witheach screen.

[0003] High throughput screening of collections of chemicallysynthesized molecules and of natural products (such as microbialfermentation broths) has thus played a central role in the search forlead compounds for the development of new pharmacological agents. Theremarkable surge of interest in combinatorial chemistry and theassociated technologies for generating and evaluating moleculardiversity represent significant milestones in the evolution of thisparadigm of drug discovery. See Pavia et al., 1993, Bioorq. Med. Chem.Lett. 3: 387-396, incorporated herein by reference. To date, peptidechemistry has been the principle vehicle for exploring the utility ofcombinatorial methods in ligand identification. See Jung &Beck-Sickinger, 1992, Angew. Chem. Int. Ed. Engl. 31: 367-383,incorporated herein by reference. This may be ascribed to theavailability of a large and structurally diverse range of amino acidmonomers, a relatively generic, high-yielding solid phase couplingchemistry and the synergy with biological approaches for generatingrecombinant peptide libraries. Moreover, the potent and specificbiological activities of many low molecular weight peptides make thesemolecules attractive starting points for therapeutic drug discovery. SeeHirschmann, 1991, Angew. Chem. Int. Ed. Engl. 30: 1278-1301, and Wiley &Rich, 1993, Med. Res. Rev. 13: 327-384, each of which is incorporatedherein by reference. Unfavorable pharmacodynamic properties such as poororal bioavailability and rapid clearance in vivo have limited the morewidespread development of peptidic compounds as drugs however. Thisrealization has recently inspired workers to extend the concepts ofcombinatorial organic synthesis beyond peptide chemistry to createlibraries of known pharmacophores like benzodiazepines (see Bunin &Ellman, 1992, J. Amer. Chem. Soc. 114: 10997-10998, incorporated hereinby reference) as well as polymeric molecules such as oligomericN-substituted glycines (“peptoids”) and oligocarbamates. See Simon etal., 1992, Proc. Natl. Acad. Sci. USA 89: 9367-9371; Zuckermann et al.,1992, J. Amer. Chem. Soc. 114: 10646-10647; and Cho et al., 1993,Science 261:1303-1305, each of which is incorporated herein byreference. In similar developments, much as modern combinatorialchemistry has resulted in a dramatic increase in the number of testcompounds that may be screened, human genome research has also uncoveredlarge numbers of new target molecules against which the efficacy of testcompounds may be screened.

[0004] Despite the improvements achieved using parallel screeningmethods and other technological advances, such as robotics and highthroughput detection systems, current screening methods still have anumber of associated problems. For example, screening large numbers ofsamples using existing parallel screening methods have high spacerequirements to accommodate the samples and equipment, e.g., robotics,etc., high costs associated with that equipment, and high reagentrequirements necessary for performing the assays. Additionally, in manycases, reaction volumes must be very small to account for the smallamounts of the test compounds that are available. Such small volumescompound errors associated with fluid handling and measurement, e.g.,evaporation. Additionally, fluid handling equipment and methods havetypically been unable to handle these volume ranges with any acceptablelevel of accuracy due in part to surface tension effects in such smallvolumes.

[0005] The development of systems to address these problems mustconsider a variety of aspects of the assay process. Such aspects includetarget and compound sources, test compound and target handling, specificassay requirements, and data acquisition, reduction storage andanalysis. In particular, there exists a need for high throughputscreening methods and associated equipment and devices that are capableof performing repeated, accurate assay screens, ancL operating at verysmall volumes.

[0006] The present invention meets these and a variety of other needs.In particular, the present invention provides novel methods andapparatuses for performing screening assays which address and providemeaningful solutions to these problems.

SUMMARY OF THE INVENTION

[0007] The present invention generally provides methods of screening aplurality of test compounds for an effect on a biochemical system. Thesemethods typically utilize microfabricated substrates which have at leasta first surface, and at least two intersecting channels fabricated intothat first surface. At least one of the intersecting channels will haveat least one cross-sectional dimension in a range from 0.1 to 500 μm.The methods involve flowing a first component of a biochemical system ina first of the at least two intersecting channels. At least a first testcompound is flowed from a second channel into the first channel wherebythe test compound contacts the first component of the. biochemicalsystem. An effect of the test compound on the biochemical system is thendetected.

[0008] In a related aspect, the method comprises continuously flowingthe first component of a biochemical system in the first channel of theat least two intersecting channels. Different test compounds areperiodically introduced into the first channel from a second channel.The effect, if any, of the test compound on the biochemical system isthen detected.

[0009] In an alternative aspect the methods utilize a substrate havingat least a first surface with a plurality of reaction channelsfabricated into the first surface. Each of the plurality of reactionchannels is fluidly connected to at least two transverse channels alsofabricated in the surface. The at least a first component of abiochemical system is introduced into the plurality of reactionchannels, and a plurality of different test compounds is flowed throughat least one of the at least two transverse channels. Further, each ofthe plurality of test compounds is introduced into the transversechannel in a discrete volume. Each of the plurality of different testcompounds is directed into a separate reaction channel and the effect ofeach of test compounds on the biochemical system is then detected.

[0010] The present invention also provides apparatuses for practicingthe above methods. In one aspect, the present invention provides anapparatus for screening test compounds for an effect on a biochemicalsystem. The device comprises a substrate having at least one surfacewith at least two intersecting channels fabricated into the surface. Theat least two intersecting channels have at least one cross-sectionaldimension in the range from about 0.1 to about 500 μm. The device alsocomprises a source of different test compounds fluidly connected to afirst of the at least two intersecting channels, and a source of atleast one component of the biochemical system fluidly connected to asecond of the at least two intersecting channels. Also included arefluid direction systems for flowing the at least one component withinthe intersecting channels, and for introducing the different testcompounds from the first to the second of the intersecting channels. Theapparatus also comprises a detection zone in the second channel fordetecting an effect of said test compound on said biochemical system.

[0011] In preferred aspects, the apparatus of the invention includes afluid direction system which comprises at least three electrodes, eachelectrode being in electrical contact with the at least two intersectingchannels on a different side of an intersection formed by the at leasttwo intersecting channels. The fluid direction system also includes acontrol system for concomitantly applying a variable voltage at each ofthe electrodes, whereby movement of the test compounds or the at leastfirst component in the at least two intersecting channels may becontrolled.

[0012] In another aspect, the present invention provides an apparatusfor detecting an effect of a test compound on a biochemical system,comprising a substrate having at least one surface with a plurality ofreaction channels fabricated into the surface. The apparatus also has atleast two transverse channels fabricated into the surface, wherein eachof the plurality of reaction channels is fluidly connected to a first ofthe at least two transverse channels at a first point in each of thereaction channels, and fluidly connected to a second transverse channelat a second point in each of the reaction channels. The apparatusfurther includes a source of at least one component of the biochemicalsystem fluidly connected to each of the reaction channels, a source oftest compounds fluidly connected to the first of the transversechannels, and a fluid direction system for controlling movement of thetest compound and the first component within the transverse channels andthe plurality reaction channels. As above, the apparatuses also includea detection zone in the second transverse channel for detecting aneffect of the test compound on the biochemical system.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a schematic illustration of one embodiment of amicrolaboratory screening assay system of the present invention whichcan be used in running a continuous flow assay system.

[0014]FIGS. 2A and 2B show a schematic illustration of the apparatusshown in FIG. 1, operating in alternate assay systems. FIG. 2A shows asystem used for screening effectors of an enzyme-substrate interaction.FIG. 2B illustrates the use of the apparatus in screening effectors ofreceptor-ligand interactions.

[0015]FIG. 3 is a schematic illustration of a “serial input parallelreaction” microlaboratory assay system in which compounds to be screenedare serially introduced into the device but then screened in a parallelorientation within the device.

[0016] FIGS. 4A-4F show a schematic illustration of the operation of thedevice shown in FIG. 3, in screening a plurality of bead based testcompounds.

[0017]FIG. 5 shows a schematic illustration of a continuous flow assaydevice incorporating a sample shunt for performing prolonged incubationfollowed by a separation step.

[0018]FIG. 6A shows a schematic illustration of a serial input parallelreaction device for use with fluid based test compounds. FIGS. 6B and 6Cshow a schematic illustration of fluid flow patterns within the deviceshown in FIG. 6A.

[0019]FIG. 7 shows a schematic illustration of one embodiment of anoverall assay systems which employs multiple microlaboratory deviceslabeled as “LabChips” for screening test compounds.

[0020]FIG. 8 illustrates the parameters of a fluid flow system on asmall chip device for performing enzyme inhibitor screening.

[0021]FIG. 9 shows a schematic illustration of timing for sample/spacerloading in a microfluidic device channel.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0022] I. General

[0023] The present invention provides novel microlaboratory systems andmethods that are useful for performing high-throughput screening assays.In particular, the present invention provides microfluidic devices andmethods of using such devices that are useful in screening large numbersof different compounds for their effects on a variety of chemical, andpreferably, biochemical systems.

[0024] As used herein, the phrase “biochemical system” generally refersto a chemical interaction that involves molecules of the type generallyfound within living organisms. Such interactions include the full rangeof catabolic and anabolic reactions which occur in living systemsincluding enzymatic, binding, signalling and other reactions. Further,biochemical systems, as defined herein, will also include model systemswhich are mimetic of a particular biochemical interaction. Examples ofbiochemical systems of particular interest in practicing the presentinvention include, e.g., receptor-ligand interactions, enzyme-substrateinteractions, cellular signaling pathways, transport reactions involvingmodel barrier systems (e.g., cells or membrane fractions) forbioavailability screening, and a variety of other general systems.Cellular or organismal viability or activity may also be screened usingthe methods and apparatuses of the present invention, i.e., intoxicology studies.

[0025] In order to provide methods and devices for screening compoundsfor effects on biochemical systems, the present invention generallyincorporates model in vitro systems which mimic a given biochemicalsystem in vivo for which effector compounds are desired. The range ofsystems against which compounds can be screened and for which effectorcompounds are desired, is extensive. For example, compounds may bescreened for effects in blocking, slowing or otherwise inhibiting keyevents associated with biochemical systems whose effect is undesirable.For example, test compounds may be screened for their ability to blocksystems that are responsible, at least in part, for the onset of diseaseor for the occurrence of particular symptoms of diseases, including,e.g., hereditary diseases, cancer, bacterial or viral infections and thelike. Compounds which show promising results in these screening assaymethods can then be subjected to further testing to identify effectivepharmacological agents for the treatment of disease or symptoms of adisease.

[0026] Alternatively, compounds can be screened for their ability tostimulate, enhance or otherwise induce biochemical systems whosefunction is believed to be desirable, e.g., to remedy existingdeficiencies in a patient.

[0027] Once a model system is selected, batteries of test compounds canthen be applied against these model systems. By identifying those testcompounds that have an effect on the particular biochemical system, invitro, one can identify potential effectors of that system, in vivo.

[0028] In their simplest forms, the biochemical system models employedin the methods and apparatuses of the present invention will screen foran effect of a test compound on an interaction between two components ofa biochemical system, e.g., receptor-ligand interaction,enzyme-substrate interaction, and the like. In this form, thebiochemical system model will typically include the two normallyinteracting components of the system for which an effector is sought,e.g., the receptor and its ligand or the enzyme and its substrate.

[0029] Determining whether a test compound has an effect on thisinteraction then involves contacting the system with the test compoundand assaying for the functioning of the system, e.g., receptor-ligandbinding or substrate turnover. The assayed function is then compared toa control, e.g., the same reaction in the absence of the test compoundor in the presence of a known effector.

[0030] Although described in terms of two-component biochemical systems,the methods and apparatuses may also be used to screen for effectors ofmuch more complex systems where the result or end product of the systemis known and assayable at some level, e.g., enzymatic pathways, cellsignaling pathways and the like. Alternatively, the methods andapparatuses described herein may be used to screen for compounds thatinteract with a single component of a biochemical system, e.g.,compounds that specifically bind to a particular biochemical compound,e.g., a receptor, ligand, enzyme, nucleic acid, structuralmacromolecule, etc.

[0031] Biochemical system models may also be embodied in whole cellsystems. For example, where one is seeking to screen test compounds foran effect on a cellular response, whole cells may be utilized. Modifiedcell systems may also be employed in the screening systems encompassedherein. For example, chimeric reporter systems may be employed asindicators of an effect of a test compound on a particular biochemicalsystem. Chimeric reporter systems typically incorporate a heterogenousreporter system integrated into a signaling pathway which signals thebinding of a receptor to its ligand. For example, a receptor may befused to a heterologous protein, e.g., an enzyme whose activity isreadily assayable. Activation of the receptor by ligand binding thenactivates the heterologous protein which then allows for detection.Thus, the surrogate reporter system produces an event or signal which isreadily detectable, thereby providing an assay for receptor/ligandbinding. Examples of such chimeric reporter systems have been previouslydescribed in the art.

[0032] Additionally, where one is screening for bioavailability, e.g.,transport, biological barriers may be included. The term “biologicalbarriers” generally refers to cellular or membranous layers withinbiological systems, or synthetic models thereof. Examples of suchbiological barriers include the epithelial and endothelial layers, e.g.vascular endothelia and the like.

[0033] Biological responses are often triggered and/or controlled by thebinding of a receptor to its ligand. For example, interaction of growthfactors, i.e., EGF, FGF, PDGF, etc., with their receptors stimulates awide variety of biological responses including, e.g., cell proliferationand differentiation, activation of mediating enzymes;, stimulation ofmessenger turnover, alterations in ion fluxes, activation of enzymes,changes in cell shape and the alteration in genetic expression levels.Accordingly, control of the interaction of the receptor and its ligandmay offer control of the biological responses caused by thatinteraction.

[0034] Accordingly, in one aspect, the present invention will be usefulin screening for compounds that affect an interaction between a receptormolecule and its ligands. As used herein, the term “receptor” generallyrefers to one member of a pair of compounds which specifically recognizeand bind to each other. The other member of the pair is termed a“ligand.” Thus, a receptor/ligand pair may include a typical proteinreceptor, usually membrane associated, and its natural ligand, e.g.,another protein or small molecule. Receptor/ligand pairs may alsoinclude antibody/antigen binding pairs, complementary nucleic acids,nucleic acid associating proteins and their nucleic acid ligands. Alarge number of specifically associating biochemical compounds are wellknown in the art and can be utilized in practicing the presentinvention.

[0035] Traditionally, methods for screening for effectors of areceptor/ligand interaction have involved incubating a receptor/ligandbinding pair in the presence of a test compound. The level of binding ofthe receptor/ligand pair is then compared to negative and/or positivecontrols. Where a decrease in normal binding is seen, the test compoundis determined to be an inhibitor of the receptor/ligand binding. Wherean increase in that binding is seen, the test compound is determined tobe an enhancer or inducer of the interaction.

[0036] In the interest of efficiency, screening assays have typicallybeen set up in multiwell reaction plates, e.g., multi-well microplates,which allow for the simultaneous, parallel screening of large numbers oftest compounds.

[0037] A similar, and perhaps overlapping, set of biochemical systemsincludes the interactions between enzymes and their substrates. The term“enzyme” as used herein, generally refers to a protein which acts as acatalyst to induce a chemical change in other compounds or “substrates.”

[0038] Typically, effectors of an enzyme's activity toward its substrateare screened by contacting the enzyme with a substrate in the presenceand absence of the compound to be screened and under conditions optimalfor detecting changes in the enzyme's activity. After a set time forreaction, the mixture is assayed for the presence of reaction productsor a decrease in the amount of substrate. The amount of substrate thathas been catalyzed is them compared to a control, i.e., enzyme contactedwith substrate in the absence of test compound or presence of a knowneffector. As above, a compound that reduces the enzymes activity towardits substrate is termed an “inhibitor,” whereas a compound thataccentuates that activity is termed an “inducer.”

[0039] Generally, the various screening methods encompassed by thepresent invention involve the serial introduction of a plurality of testcompounds into a microfluidic device. Once injected into the device, thetest compound may be screened for effect on a biological system using acontinuous serial or parallel assay orientation.

[0040] As used herein, the term “test compound” refers to the collectionof compounds that are to be screened for their ability to affect aparticular biochemical system. Test compounds may include a wide varietyof different compounds, including chemical compounds, mixtures ofchemical compounds, e.g., polysaccharides, small organic or inorganicmolecules, biological macromolecules, e.g., peptides, proteins, nucleicacids, or an extract made from biological materials such as bacteria,plants, fungi, or animal cells or tissues, naturally occurring orsynthetic compositions. Depending upon the particular embodiment beingpracticed, the test compounds may be provided, e.g., injected, free insolution, or may be attached to a carrier, or a solid support, e.g.,beads. A number of suitable solid supports may be employed forimmobilization of the test compounds. Examples of suitable solidsupports include agarose, cellulose, dextran (commercially available as,i.e., Sephadex, Sepharose) carboxymethyl cellulose, polystyrene,polyethylene glycol (PEG), filter paper, nitrocellulose, ion exchangeresins, plastic films, glass beads, polyaminemethylvinylether maleicacid copolymer, amino acid copolymer, ethylene-maleic acid copolymer,nylon, silk, etc. Additionally, for the methods and apparatusesdescribed herein, test compounds may be screened individually, or ingroups. Group screening is particularly useful where hit rates foreffective test compounds are expected to be low such that one would notexpect more than one positive result for a given group.

[0041] II. Assay Systems

[0042] As described above, the screening methods of the presentinvention are generally carried out in microfluidic devices or“microlaboratory systems,” which allow for integration of the elementsrequired for performing the assay, automation, and minimal environmentaleffects on the assay system, e.g., evaporation, contamination, humanerror. A number of devices for carrying out the assay methods of theinvention are described in substantial detail below. However, it will berecognized that the specific configuration of these devices willgenerally vary depending upon the type of assay and/or assay orientationdesired. For example, in some embodiments, the screening methods of theinvention can be carried out using a microfluidic device having twointersecting channels. For more complex assays or assay orientations,multichannel/intersection devices may be employed. The small scale,integratability and self-contained nature of these devices allows forvirtually any assay orientation to be realized within the context of themicrolaboratory system.

[0043] A. Continuous Flow Assay Systems

[0044] In one preferred aspect, the methods and apparatuses of theinvention are used in screening test compounds using a continuous flowassay system. Generally, the continuous flow assay system can be readilyused in screening for inhibitors or inducers of enzymatic activity, orfor agonists or antagonists of receptor-ligand binding. In brief, thecontinuous flow assay system involves the continuous flow of theparticular biochemical system along a microfabricated channel. As usedherein, the term “continuous” generally refers to an unbroken orcontiguous stream of the particular composition that is beingcontinuously flowed. For example, a continuous flow may include aconstant fluid flow having a set velocity, or alternatively, a fluidflow which includes pauses in the flow rate of the overall system, suchthat the pause does not otherwise interupt the flow stream. Thefunctioning of the system is indicated by the production of a detectableevent or signal. Typically, such detectable signals will includechromophoric or fluorescent signals that are associated with thefunctioning of the particular model system used. For enzyme systems,such signals will generally be produced by products of the enzyme'scatalytic action, e.g., on a chromogenic or fluorogenic substrate. Forbinding systems, e.g., receptor ligand interactions, signals willtypically involve the association of a labeled ligand with the receptor,or vice versa.

[0045] In preferred aspects, the continuous system generates a constantsignal which varies only when a test compound is introduced that affectsthe system. Specifically, as the system components flow along thechannel, they will produce a relatively constant signal level at adetection zone or window of the channel. Test compounds are periodicallyintroduced into the channel and mixed with the system components. Wherethose test compounds have an effect on the system, it will cause adeviation from the constant signal level at the detection window. Thisdeviation may then be correlated to the particular test compoundscreened.

[0046] One embodiment of a device for use in a serial or continuousassay geometry is shown in FIG. 1. As shown, the overall device 100 isfabricated in a planar substrate 102. Suitable substrate materials aregenerally selected based upon their compatibility with the conditionspresent in the particular operation to be performed by the device. Suchconditions can include extremes of pH, temperature, salt concentration,and application of electrical fields. Additionally, substrate materialsare also selected for their inertness to critical components of ananalysis or synthesis to be carried out by the device.

[0047] Examples of useful substrate materials include, e.g., glass,quartz and silicon as well as polymeric substrates, e.g. plastics. Inthe case of conductive or semi-conductive substrates, it will generallybe desirable to include an insulating layer on the substrate. This isparticularly important where the device incorporates electricalelements, e.g., electrical fluid direction systems, sensors and thelike. In the case of polymeric substrates, the substrate materials maybe rigid, semi-rigid, or non-rigid, opaque, semi-opaque or transparent,depending upon the use for which they are intended. For example, deviceswhich include an optical or visual detection element, will generally befabricated, at least in part, from transparent materials to allow, or atleast, facilitate that detection. Alternatively, transparent windows of,e.g., glass or quartz, may be incorporated into the device for thesetypes detection elements. Additionally, the polymeric materials may havelinear or branched backbones, and may be crosslinked or non-crosslinked.Examples of particularly preferred polymeric materials include, e.g.,polydimethylsiloxanes (PDMS), polyurethane, polyvinylchloride (PVC)polystyrene, polysulfone, polycarbonate and the like.

[0048] The device shown in FIG. 1 includes a series of channels 110,112, and optional reagent channel 114, fabricated into the surface ofthe substrate. At least one of these channels will typically have verysmall cross sectional dimensions, e.g., in the range of from about 0.1μm to about 500 μm. Preferably the cross-sectional dimensions of thechannels will be in the range of from about 0.1 to about 200 μm and morepreferably in the range of from about 0.1 to about 100 μm. Inparticularly preferred aspects, each of the channels will have at leastone cross-sectional dimension in the range of from about 0.1 μm to about100 μm. Although generally shown as straight channels, it will beappreciated that in order to maximize the use of space on a substrate,serpentine, saw tooth or other channel geometries, to incorporateeffectively longer channels in shorter distances.

[0049] Manufacturing of these microscale elements into the surface ofthe substrates may generally be carried out by any number ofmicrofabrication techniques that are well known in the art. For example,lithographic techniques may be employed in fabricating, e.g., glass,quartz or silicon substrates, using methods well known in thesemiconductor manufacturing industries such as photolithographicetching, plasma etching or wet chemical etching. Alternatively,micromachining methods such as laser drilling, micromilling and the likemay be employed. Similarly, for polymeric substrates, well knownmanufacturing techniques may also be used. These techniques includeinjection molding or stamp molding methods where large numbers ofsubstrates may be produced using, e.g., rolling stamps to produce largesheets of microscale substrates or polymer microcasting techniques wherethe substrate is polymerized within a micromachined mold.

[0050] The devices will typically include an additional planar elementwhich overlays the channeled substrate enclosing and fluidly sealing thevarious channels to form conduits. Attaching the planar cover elementmay be achieved by a variety of means, including, e.g., thermal bonding,adhesives or, in the case of certain substrates, e.g., glass, orsemi-rigid and non-rigid polymeric substrates, a natural adhesionbetween the two components. The planar cover element may additionally beprovided with access ports and/or reservoirs for introducing the variousfluid elements needed for a particular screen.

[0051] The device shown in FIG. 1 also includes reservoirs 104, 106 and108, disposed and fluidly connected at the ends of the channels 110 and114. As shown, sample channel 112, is used to introduce the plurality ofdifferent test compounds into the device. As such, this channel willgenerally be fluidly connected to a source of large numbers of separatetest compounds that will be individually introduced into the samplechannel 112 and subsequently into channel 110.

[0052] The introduction of large numbers of individual, discrete volumesof test compounds into the sample may be carried out by a number ofmethods. For example, micropipettors may be used to introduce the testcompounds into the device. In preferred aspects, an electropipettor maybe used which is fluidly connected to sample channel 112. An example ofsuch an electropipettor is described in, e.g., U.S. patent applicationSer. No. ______, filed ______ (Attorney Docket No. 017646-000500) thedisclosure of which is hereby incorporated herein by reference in itsentirety for all purposes. Generally, this electropipettor utilizeselectroosmotic fluid direction as described herein, to alternatelysample a number of test compounds and spacer compounds. The pipettorthen delivers individual, physically isolated sample or test compoundvolumes, in series, into the sample channel for subsequent manipulationwithin the device. Individual samples are typically separated by a slugof low ionic strength spacer fluid. These low ionic strength spacershave higher voltage drop over the length of the plug, thereby drivingthe electrokinetic pumping. On either side of the sample plug, which istypically in higher ionic strength solution, are fluid plugs referred toas guard plugs or bands at the interface of the sample plug. These guardbands typically comprise a high ionic strength solution to preventmigration of the sample elements into the spacer fluid band, resultingin electrophoretic bias. The use of such guard bands is described ingreater detail in U.S. patent application Ser. No. ______, filed ______,(Attorney Docket No. 017646-000500) which is incorporated herein byreference.

[0053] Alternatively, the sample channel 112 may be individually fluidlyconnected to a plurality of separate reservoirs via separate channels.The separate reservoirs each contain a separate test compound withadditional reservoirs being provided for appropriate spacer compounds.The test compounds and/or spacer compounds are then transported from thevarious reservoirs into the sample channels using appropriate fluiddirection schemes. In either case, it generally is desirable to separatethe discrete sample volumes, or test compounds, with an appropriatespacer buffer.

[0054] As shown, the device also includes a Detection window or zone 116at which a signal from the biochemical system may be monitored. Thisdetection window typically will include a transparent cover allowingvisual or optical observation and detection of the assay results, e.g.,observation of a colorometric or fluorometric response.

[0055] In particularly preferred aspects, monitoring of the signals atthe detection window is achieved using an optical detection system. Forexample, fluorescence based signals are typically monitored using, e.g.,laser activated fluorescence detection systems which employ a laserlight source at an appropriate wavelength for activating the fluorescentindicator within the system. Fluorescence is then detected using anappropriate detector element, e.g., a photomultiplier tube (PMT).Similarly, for screens employing colorometric signals,spectrophotometric detection systems may be employed which direct alight source at the sample and provide a measurement of absorbance ortransmissivity of the sample.

[0056] In alternative aspects, the detection system may comprise anon-optical detectors or sensors for detecting a particularcharacteristic of the system disposed within detection window 116. Suchsensors may include temperature, conductivity, potentiometric (pH,ions), amperometric (for compounds that may be oxidized or reduced,e.g., O₂, H₂O₂, I₂, oidzable/reducible organic compounds, and the like).

[0057] In operation, a fluid first component of a biological system,e.g., a receptor or enzyme, is placed in reservoir 104. The firstcomponent is flowed through main channel 110, past the detection window,116, and toward waste reservoir 108. A second component of thebiochemical system, e.g., a ligand or substrate, is concurrently flowedinto the main channel 110 from the side channel 114, whereupon the firstand second components mix and are able to interact. Deposition of theseelements within the device may be carried out in a number of ways. Forexample, the enzyme and substrate, or receptor and ligand solutions canbe introduced into the device through sealable access ports in theplanar cover. Alternatively, these components may be added to theirrespective reservoirs during manufacture of the device. In the case ofsuch pre-added components, it may be desirable to provide thesecomponents in a stabilized form to allow for prolonged shelf-life of thedevice. For example, the enzyme/substrate or receptor/ligand componentsmay be provided within the device in lyophilized form. Prior to use,these components may be easily reconstituted by introducing a buffersolution into the reservoirs. Alternatively, the components may belyophilized with appropriate buffering salts, whereby simple wateraddition is all that is required for reconstitution.

[0058] As noted above, the interaction of the first and secondcomponents is typically accompanied by a detectable signal. For example,in those embodiments where the first component is an enzyme and thesecond a substrate, the substrate may be a chromogenic or fluorogenicsubstrate which produces an optically detectable signal when the enzymeacts upon the substrate. In the case where the first component is areceptor and the second is a ligand, either the ligand or the receptormay bear a detectable signal. In either event, the mixture and flow rateof compounds will typically remain constant such that the flow of themixture of the first and second components past the detection window 116will produce a steady-state signal. By “steady state signal” isgenerally meant a signal that has a regular, predictable signalintensity proile. As such, the steady-state signal may include signalshaving a constant signal intensity, or alternatively, a signal with aregular periodic intensity, against which variations in the normalsignal profile may be measured. This latter signal may be generated incases where fluid flow is periodically interrupted for, e.g., loadingadditional test compounds, as described in the description of thecontinuous flow systems. Although the signal produced in theabove-described enzymatic system will vary along the length of thechannel, i.e., increasing with time of exposure as the enzyme convertsthe fluorogenic substrate to the fluorescent product, the signal at anyspecific point along the channel will remain constant, given a constantflow rate.

[0059] From sample channel 112, test compounds may be periodically orserially introduced into the main channel 110 and into the stream offirst and second components. Where these test compounds have an effecton the interaction of the first and second elements, it will produce adeviation in the signal detected at the detection window. As notedabove, typically, the various different test compounds to be injectedthrough channel 112 will be separated by a spacer fluid to allowdifferentiation of the effects, or lack of effects, from one testcompound to another. In those embodiments where electroosmotic fluiddirection systems are employed, the spacer fluids may also function toreduce any electrophoretic bias that can occur within the test sample.The use of these spacer fluids as well as the general elimination ofelectrophoretic bias within the sample or test compound plugs issubstantially described in U.S. patent application Ser. No. ______,filed ______ (Attorney Docket No. 017646-000500) previously incorporatedherein by reference.

[0060] By way of example, a steady, continuous flow of enzyme andfluorogenic substrate through main channel 110 will produce a constantfluorescent signal at the detection window 116. Where a test compoundinhibits the enzyme, it will produce a momentary but detectable drop inthe Level of signal at the detection window. The timing of the drop insignal can then be correlated with a particular test compound based upona known injection to detection time-frame. Specifically, the timerequired for an injected compound to produce an observed effect can bereadily determined using positive controls.

[0061] For receptor/ligand systems, a similar variation in the steadystate signal may also be observed. Specifically, the receptor and itsfluorescent ligand can be made to have different flow rates along thechannel. This can be accomplished by incorporating size exclusionmatrices within the channel, or, in the case of electroosmotic methods,altering the relative electrophoretic mobility of the two compounds sothat the receptor flows more rapidly down the channel. Again, this maybe accomplished through the use of size exclusion matrices, or throughthe use of different surface charges in the channel which will result indifferential flow rates of charge-varied compounds. Where a testcompound binds to the receptor, it will result in a dark pulse in thefluorescent signal followed by a brighter pulse. Without being bound toa particular theory of operation, it is believed that the steady statesignal is a result of both free fluorescent ligand, and fluorescentligand bound to the receptor. The bound ligand is traveling at the sameflow rate as the receptor while the unbound ligand is traveling moreslowly. Where the test compound inhibits the receptor-ligandinteraction, the receptor will not ‘bring along’ the fluorescent ligand,thereby diluting the fluorescent ligand in the direction of flow, andleaving an excess of free fluorescent ligand behind. This results in atemporary reduction in the steady-state signal, followed by a temporaryincrease in fluorescence. Alternatively, schemes similar to thoseemployed for the enzymatic system may be employed, where there is asignal that reflects the interaction of the receptor with its ligand.For example, pH indicators which indicate pH effects of receptor-ligandbinding may be incorporated into the device along with the biochemicalsystem, i.e., in the form of encapsulated cells, whereby slight pHchanges resulting from binding can be detected. See Weaver, et al.,Bio/Technology (1988) 6:1084-1089. Additionally, one can monitoractivation of enzymes resulting from receptor ligand binding, e.g.,activation of kinases, or detect conformational changes in such enzymesupon activation, e.g., through incorporation of a fluorophore which isactuivated or quenched by the conformational change to the enzyme uponactivation.

[0062] Flowing and direction of fluids within the microscale fluidicdevices may be carried out by a variety of methods. For example, thedevices may include integrated microfluidic structures, such asmicropumps and microvalves, or external elements, e.g., pumps andswitching valves, for the pumping and direction of the various fluidsthrough the device. Examples of microfluidic structures are describedin, e.g., U.S. Pat. Nos. 5,271,724, 5,277,556, 5,171,132, and 5,375,979.See also, Published U.K. Patent Application No. 2 248 891 and PublishedEuropean Patent Application No. 568 902.

[0063] Although microfabricated fluid pumping and valving systems may bereadily employed in the devices of the invention, the cost andcomplexity associated with their manufacture and operation can generallyprohibit their use in mass-produced disposable devices as are envisionedby the present invention. For that reason, in particularly preferredaspects, the devices of the invention will typically include anelectroosmotic fluid direction system. Such fluid direction systemscombine the elegance a fluid direction system devoid of moving parts,with an ease of manufacturing, fluid control and disposability. Examplesof particularly preferred electroosmotic fluid direction systemsinclude, e.g., those described in International Patent Application No.Wo 96/04547 to Ramsey et al., which is incorporated herein by referencein its entirety for all purposes.

[0064] In brief, these fluidic control systems typically includeelectrodes disposed within the reservoirs that are placed in fluidconnection with the plurality of intersecting channels fabricated intothe surface of the substrate. The materials stored in the reservoirs aretransported through the channel system delivering appropriate volumes ofthe various materials to one or more regions on the substrate in orderto carry out a desired screening assay.

[0065] Fluid transport and direction is accomplished throughelectroosmosis or electrokinesis. In brief, when an appropriate fluid isplaced in a channel or other fluid conduit having functional groupspresent at the surface, those groups can ionize. For example, where thesurface of the channel includes hydroxyl functional groups at thesurface, protons can leave the surface of the channel and enter thefluid. Under such conditions, the surface will possess a net negativecharge, whereas the fluid will possess an excess of protons or positivecharge, particularly localized near the interface between the channelsurface and the fluid. By applying an electric field along the length ofthe channel, cations will flow toward the negative electrode. Movementof the positively charged species in the fluid pulls the solvent withthem. The steady state velocity of this fluid movement is generallygiven by the equation:$v = \frac{\varepsilon \quad \xi \quad E}{4{\pi\eta}}$

[0066] where v is the solvent velocity, ε is the dielectric constant ofthe fluid, ξ is the zeta potential of the surface, E is the electricfield strength, and η is the solvent viscosity. Thus, as can be easilyseen from this equation, the solvent velocity is directly proportionalto the surface potential.

[0067] To provide appropriate electric fields, the system generallyincludes a voltage controller that is capable of applying selectablevoltage levels, simultaneously, to each of the reservoirs, includingground. Such a voltage controller can be implemented using multiplevoltage dividers and multiple relays to obtain the selectable voltagelevels. Alternatively, multiple, independent voltage sources may beused. The voltage controller is electrically connected to each of thereservoirs via an electrode positioned or fabricated within each of theplurality of reservoirs.

[0068] Incorporating this electroosmotic fluid direction system into thedevice shown in FIG. 1 involves incorporation of an electrode withineach of the reservoirs 104, 106 and 108, and at the terminus of samplechannel 112 or at the terminus of any fluid channels connected thereto,whereby the electrode is in electrical contact with the fluid disposedin the respective reservoir or channel. Substrate materials are alsoselected to produce channels having a desired surface charge. In thecase of glass substrates, the etched channels will possess a netnegative charge resulting from the ionized hydroxyls naturally presentat the surface. Alternatively, surface modifications may be employed toprovide an appropriate surface charge, e.g., coatings, derivatization,e.g., silanation, or impregnation of the surface to provideappropriately charged groups on the surface. Examples of such treatmentsare described in, e.g., Provisional Patent Application Serial No.______, filed Apr. 16, 1996 (Attorney Docket No. 017646-002600) which ishereby incorporated herein by reference in its entirety for allpurposes.

[0069] Modulating voltages are then concomitantly applied to the variousreservoirs to affect a desired fluid flow characteristic, e.g.,continuous flow of receptor/enzyme, ligand/substrate toward the wastereservoir with the periodic introduction of test compounds.Particularly, modulation of the voltages applied at the variousreservoirs can move and direct fluid flow through the interconnectedchannel structure of the device in a controlled manner to effect thefluid flow for the desired screeening assay and apparatus.

[0070]FIG. 2A shows a schematic illustration of fluid direction during atypical assay screen. Specifically, shown is the injection of a testcompound into a continuous stream of an enzyme-fluorogenic substratemixture. As shown in FIG. 2A, and with reference to FIG. 1, a continuousstream of enzyme is flowed from reservoir 104, along main channel 110.Test compounds 120, separated by appropriate fluid spacers 121 areintroduced from sample channel 112 into main channel 110. Onceintroduced into the main channel, the test compounds will interact withthe flowing enzyme stream. The mixed enzyme/test compound plugs are thenflowed along main channel 110 past the intersection with channel 114. Acontinuous stream of fluorogenic or chromogenic substrate which iscontained in reservoir 106, is introduced into sample channel 110,whereupon it contacts and mixes with the continuous stream of enzyme,including the discrete portions (or “plugs”) of the stream which includethe test compounds 122. Action of the enzyme upon the substrate willproduce an increasing level of the fluorescent or chromatic signal. Thisincreasing signal is indicated by the increasing shading within the mainchannel as it approaches the detection window. This signal trend willalso occur within those test compound plugs which have no effect on theenzyme/substrate interaction, e.g., test compound 126. Where a testcompound does have an effect on the interaction of the enzyme and thesubstrate, a variation will appear in the signal produced. For example,assuming a fluorogenic substrate, a test compound which inhibits theinteraction of the enzyme with its substrate will result in lessfluorescent product being produced within that plug. This will result ina non-fluorescent, or detectably less fluorescent, plug within theflowing stream as it passes detection window 116. For example, as shown,test compound 128, a putative inhibitor of the enzyme-substrateinteraction, shows detectably lower flourescence than the surroundingstream. This is indicated by a lack of shading of test compound plug128.

[0071] A detector adjacent to the detection window monitors the level offluorescent signal being produced by the enzyme's activity on thefluorogenic or chromogenic substrate. This signal remains at arelatively constant level for those test compounds which have no effecton the enzyme-substrate interaction. When an inhibitory compound isscreened, however, it will produce a momentary drop in the fluorescentsignal representing the reduced or inhibited enzyme activity toward thesubstrate. Conversely, inducer compounds upon screening, will produce amomentary increase in the fluorescent signal, corresponding to theincreased enzyme activity toward the substrate.

[0072]FIG. 2B provides a similar schematic illustration of a screen foreffectors of a receptor-ligand interaction. As in FIG. 2A, a continuousstream of receptor is flowed from reservoir 104 through main channel110. Test compounds 150 separated by appropriate spacer fluids 121 areintroduced into the main channel 110 from sample channel 112, and acontinuous stream of fluorescent ligand from reservoir 106 is introducedfrom side channel 114. Fluorescence is indicated by shading within thechannel. As in FIG. 2A, the continuous stream of fluorescent ligand andreceptor past the detection window 116 will provide a constant signalintensity. The portions of the stream containing the test compoundswhich have no effect on the receptor-ligand interaction will provide thesame or similar level of fluorescence as the rest of the surroundingstream, e.g., test compound 152. However, the presence of test compoundswhich possess antagonistic or inhibitory activity toward thereceptor-ligand interaction will result in lower levels of thatinteraction in those portions of the stream where those compounds arelocated, e.g., test compound 154. Further, differential flow rates forthe receptor bound fluorescent ligand and free fluorescent ligand willresult in a detectable drop in the level of fluorescence whichcorresponds to the dilution of the fluorescence resulting from unbound,faster moving receptor. The drop in fluorescence is then followed by anincrease in fluorescence 156 which corresponds to an accumulation of theslower moving, unbound fluorescent ligand.

[0073] In some cases, it may be desirable to provide an additionalchannel for shunting off or extracting the reaction mixture slug fromthe running buffer and/or spacer compounds. This may be the case whereone wishes to keep the reaction elements contained within the sampleplug during the reaction, while allowing these elements to be separatedduring a data aquisition stage. As described previously, one can keepthe various elements of the reaction together in the sample plug that ismoving through the reaction channel by incorporating appropriate spacerfluids between samples. Such spacer fluids are generally selected toretain the samples within their original slugs, i.e., not allowingsmearing of the sample into the spacer fluid, even during prolongedreaction periods. However, this goal can be at odds with those assayswhich are based upon the separation of elements of the assay, e.g.,ligand-receptor assays described above, or where a reaction product mustbe separated in a capillary.

[0074] A schematic illustration of one embodiment of a device 500 forperforming this sample shunting or extraction is shown in FIG. 5. Asshown, the samples or test compounds 504 are introduced to the device orchip via the sample channel 512. Again, these are typically introducedvia an appropriate sample injection device 506, e.g., a capillarypipettor. The ionic strength and lengths of the spacer solution plugs502 and guard band plugs 508 are selected such that those samples withthe highest electrophoretic mobility will not migrate through the spacerfluid/guard bands in the length of time that it takes the sample totravel down the reaction channel.

[0075] Assuming a receptor ligand assay system, test compounds pass intothe device 500 and into reaction channel 510, where they are firstcombined with the receptor. The test compound/receptor are flowed alongthe reaction channel in the incubation zone 510 a. Following thisinitial incubation, the test compound/receptor mix is combined with alabelled ligand (e.g., fluorescent ligand) whereupon this mixture flowsalong the second incubation region 510 b of reaction channel 510. Thelengths of the incubation regions and the flow rates of the system(determined by the potentials applied at each of the reservoirs 514,516, 518, 520, 522, and at the terminus of sample channel 512) determinethe time of incubation of the receptor with the fluorescent ligand andtest compound. The ionic strengths of the solutions containing thereceptors and fluorescent ligands, as well as the flow rates of materialfrom the reservoirs housing these elements into the sample channel areselected so as to not interfere with the spacer fluid/guard bands.

[0076] The isolated sample plugs containing receptor, fluorescent ligandand test compound are flowed along the reaction channel 510 by theapplication of potentials at, e.g., reservoirs 514, 516, 518 and at theterminus of sample channel 512. Potentials are also applied atreservoirs 520 and 522, at the opposite ends of separation channel 524,to match the potentials at the two ends of the transfer channel, so thatthe net flow across the transfer channel is zero. As the sample plugpasses the intersection of reaction channel 510 and transfer channel526, the potentials are allowed to float at reservoirs 518 and 522,whereupon the potentials applied at reservoirs 514, 516, 520, and at theterminus of sample channel 512, result in the sample plug being shuntedthrough transfer channel 526 and into separation channel 524. Once inthe separation channel, the original potentials are reapplied to all ofthe reservoirs to stop the net fluid flow through transfer channel 526.The diversion of the sample plugs can then be repeated with eachsubsequent sample plug. Within the separation channel, the sample plugmay be exposed to different conditions than those of the reactionchannel. For example, a different flow rate may be used, capillarytreatments may allow for separation of differentially charged ordifferent sized species, and the like. In a preferred aspect, samplesare shunted into the separation channel to place the samples into acapillary filled with high ionic strength buffer, i.e., to remove thelow ionic strength spacer, thereby allowing separation of the varioussample components outside the confines of the original sample plug. Forexample, in the case of the above-described receptor/ligand screen, thereceptor/ligand complex may have a different electrophoretic mobilityfrom the ligand alone, in the transfer channel, thereby allowing morepronounced separation of the complex from the ligand, and its subsequentdetection.

[0077] Such modifications have a wide variety of uses, particularlywhere it may be desirable to separate reaction products followingreaction, e.g., in cleavage reactions, fragmentation reactions, PCRreactions, and the like.

[0078] B. Serial in Parallel Assay Systems

[0079] More complex systems can also be produced within the scope of thepresent invention. For example, Et schematic illustration of onealternate embodiment employing a “serial input parallel reaction”geometry is shown in FIG. 3. As shown, the device 300 again includes aplanar substrate 302 as described previously. Fabricated into thesurface of the substrate 302 are a series of parallel reaction channels312-324. Also shown are three transverse channels fluidly connected toeach of these parallel reaction channels. The three transverse channelsinclude a sample injection channel 304, an optional seeding channel 306and a collection channel 308. Again, the substrate and channels aregenerally fabricated utilizing the materials and to the dimensionsgenerally described above. Although shown and described in terms of aseries of parallel channels, the reaction channels may also befabricated in a variety of different orientations. For example, ratherthan providing a series of parallel channels fluidly connected to asingle transverse channel, the channels may be fabricated connecting toand extending radially outward from a central reservoir, or may bearranged in some other non-parallel fashion. Additionally, althoughshown with three transverse channels, it will be recognized that fewertransverse channels may be used where, e.g., the biochemical systemcomponents are predisposed within the device. Similarly, where desired,more transverse channels may be used to introduce further elements intoa given assay screen. Accordingly, the serial-in-parallel devices of thepresent invention will typically include at least two and preferablythree, four, five or more transverse channels. Similarly, although shownwith 7 reaction channels, it will be readily appreciated that themicroscale devices of the present invention will be capable ofcomprising more than 7 channels, depending upon the needs of theparticular screen. In preferred aspects, the devices will include from10 to about 500 reaction channels, and more preferably, from 20 to about200 reaction channels.

[0080] This device may be particularly useful for screening testcompounds serially injected into the device, but employing a parallelassay geometry, once the samples are introduced into the device, toallow for increased throughput.

[0081] In operation, test compounds are serially introduced into thedevice, separated as described above, and flowed along the transversesample injection channel 304 until the separate test compounds areadjacent the intersection of the sample channel 304 with the parallelreaction channels 310-324. As shown in FIGS. 4A-4F, the test compoundsmay be provided immobilized on individual beads. In those cases wherethe test compounds are immobilized on beads, the parallel channels maybe optionally fabricated to include bead resting wells 326-338 at theintersection of the reaction channels with the sample injection channel304. Arrows 340 indicate the net fluid flow during this type ofsample/bead injection. As individual beads settle into a resting well,fluid flow through that particular channel will be generally restricted.The next bead in the series following the unrestricted fluid flow, thenflows to the next available resting well to settle in place.

[0082] Once in position adjacent to the intersection of the parallelreaction channel and the sample injection channel, the test compound isdirected into its respective reaction channel by redirecting fluid flowsdown those channels. Again, in those instances where the test compoundis immobilized on a bead, the immobilization will typically be via acleavable linker group, e.g., a photolabile, acid or base labile linkergroup. Accordingly, the test compound will typically need to be releasedfrom the bead, e.g., by exposure to a releasing agent such as light,acid, base or the like prior to flowing the test compound down thereaction channel.

[0083] Within the parallel channel, the test compound will be contactedwith the biochemical system for which an effector compound is beingsought. As shown, the first component of the biochemical system isplaced into the reaction channels using a similar technique to thatdescribed for the test compounds. In particular, the particularbiochemical system is typically introduced via one or more transverseseeding channels 306. Arrows 342 illustrate the direction of fluid flowwithin the seeding channel 306. The biochemical system may be solutionbased, e.g., a continuously flowing enzyme/substrate or receptor ligandmixture like that described above, or as shown in FIGS. 4A-4F, may be awhole cell or bead based system, e.g., beads which have enzyme/substratesystems immobilized thereon.

[0084] In those instances where the biochemical system is incorporatedin a particle, e.g., a cell or bead, the parallel channel may include aparticle retention zone 344. Typically, such retention zones willinclude a particle sieving or filtration matrix, e.g., a porous gel ormicrostructure which retains particulate material but allows the freeflow of fluids. Examples of microstructures for this filtration include,e.g., those described in U.S. Pat. No. 5,304,487, which is herebyincorporated by reference in its entirety for all purposes. As with thecontinuous system, fluid direction within the more complex systems maybe generally controlled using microfabricated fluid directionstructures, e.g., pumps and valves. However, as the systems grow morecomplex, such systems become largely unmanageable. Accordingly,electroosmotic systems, as described above, are generally preferred forcontrolling fluid in these more complex systems. Typically, such systemswill incorporate electrodes within reservoirs disposed at the termini ofthe various transverse channels to control fluid flow thorough thedevice. In some aspects, it may be desirable to include electrodes atthe termini of all the various channels. This generally provides formore direct control, but also grows less managable as systems grow morecomplex. In order to utilize fewer electrodes and thus reduce thepotential complexity, it may often be desireable in parallel systems,e.g., where two fluids are desired to move at similar rates in parallelchannels, to adjust the geometries of the various flow channels. Inparticular, as channel length increases, resistance along that channelwill also increase. As such, flow lengths between electrodes should bedesigned to be substantially the same regardless of the parallel pathchosen. This will generally prevent the generation of transverseelectrical fields and thus promote equal flow in all parallel channels.To accomplish substantially the same resistance between the electrodes,one can alter the geometry of the channel structure to provide for thesame channel length, and thus, the channel resistance, regardless of thepath travelled. Alternatively, resistance of channels may be adjusted byvarying the cross-sectional dimensions of the paths, thereby creatinguniform resistance levels regardless of the path taken.

[0085] As the test compounds are drawn through their respective parallelreaction channels, they will contact the biochemical system in question.As described above, the particular biochemical system will typicallyinclude a flowable indicator system which indicates the relativefunctioning of that system, e.g., a soluble indicator such aschromogenic or fluorogenic substrate, labelled ligand, or the like, or aparticle based signal, such as a precipitate or bead bound signallinggroup. The flowable indicator is then flowed through the respectiveparallel channel and into the collection channel 308 whereupon thesignals from each of the parallel channels are flowed, in series, pastthe detection window, 116.

[0086] FIGS. 4A-4F, with reference to FIG. 3, show a schematicillustration of the progression of the injection of test compounds andbiochemical system components into the “serial input parallel reaction”device, exposure of the system to the test compounds, and flowing of theresulting signal out of the parallel reaction channels and past thedetection window. In particular, FIG. 4A shows the introduction of testcompounds immobilized on beads 346 through sample injection channel 304.Similarly, the biochemical system components 348 are introduced into thereaction channels 312-324 through seeding channel 306. Although shown asbeing introduced into the device along with the test compounds, asdescribed above, the components of the model system to be screened maybe incorporated into the reaction channels during manufacture. Again,such components may be provided in liquid form or in lyophilized formfor increased shelf life of the particular screening device.

[0087] As shown, the biochemical system components are embodied in acellular or particle based system, however, fluid components may also beused as described herein. As the particulate components flow into thereaction channels, they may be retained upon an optional particleretaining matrix 344, as described above.

[0088]FIG. 4B illustrates the release of test compounds from the beads346 by exposing the beads to a releasing agent. As shown, the beads areexposed to light from an appropriate light source 352, e.g., which isable to produce light in a wavelength sufficient to photolyze the linkergroup, thereby releasing compounds that are coupled to their respectivebeads via a photolabile linker group.

[0089] In FIG. 4C, the released test compounds are flowed into and alongthe parallel reaction channels as shown by arrows 354 until they contactthe biochemical system components. The biochemical system components 348are then allowed to perform their function, e.g., enzymatic reaction,receptor/ligand interaction, and the like, in the presence of the testcompounds. Where the various components of the biochemical system areimmobilized on a solid support, release of the components from theirsupports can provide the initiating event for the system. A solublesignal 356 which corresponds to the functioning of the biochemicalsystem is then generated (FIG. 4D). As described previously, a variationin the level of signal produced is an indication that the particulartest compound is an effector of the particular biochemical system. Thisis illustrated by the lighter shading of signal 358.

[0090] In FIGS. 4E and 4F, the soluble signal is then flowed out ofreactions channels 312-324 into the detection channel 308, and along thedetection channel past the detection window 116.

[0091] Again, a detection system as described above, located adjacentthe detection window will monitor the signal levels. In someembodiments, the beads which bore the test compounds may be recovered toidentify the test compounds which were present thereon. This istypically accomplished by incorporation of a tagging group during thesynthesis of the test compound on the bead. As shown, spent bead 360,i.e., from which a test compound has been released, may be transportedout of the channel structure through port 362 for identification of thetest compound that had been coupled to it. Such identification may beaccomplished outside of the device by directing the bead to a fractioncollector, whereupon the test compounds present on the beads may beidentified, either through identification of a tagging group, or throughidentification of residual compounds. Incorporation of tagging groups incombinatorial chemistry methods has been previously described usingencrypted nucleotide sequences or chlorinated/fluorinated aromaticcompounds as tagging groups. See, e.g., Published PCT Application No. WO95/12608. Alternatively, the beads may be transported to a separateassay system within the device itself whereupon the identification maybe carried out.

[0092]FIG. 6A shows an alternate embodiment of a “serial input parallelreaction” device which can be used for fluid based as opposed to beadbased systems. As shown the device 600 generally incorporates at leasttwo transverse channels as were shown in FIGS. 3 and 4, namely, sampleinjection channel 604 and detection channel 606. These transversechannels are interconnected by the series of parallel channels 612-620which connect sample channel 604 to detection channel 606.

[0093] The device shown also includes an additional set of channels fordirecting the flow of fluid test compounds into the reaction channels.In particular, an additional transverse pumping channel 634 is fluidlyconnected to sample channel 604 via a series of parallel pumpingchannels 636-646. The pumping channel includes reservoirs 650 and 652 atits termini. The intersections of parallel channels 636-646 arestaggered from the intersections of parallel channels 612-620 withsample channel 604, e.g., half way between. Similarly, transversepumping channel 608 is connected to detection channel 606 via parallelpumping channels 622-632. Again, the intersections of parallel pumpingchannels 622-632 with detection channel 606 are staggered from theintersections of reaction channels 612-620 with the detection channel606.

[0094] A schematic illustration of the operation of this system is shownin FIGS. 6B-6C. As shown, a series of test compounds, physicallyisolated from each other, are introduced into sample channel 604 usingthe methods described previously. For electrokinetic systems, potentialsare applied at the terminus of sample channel 604, as well as reservoir648. Potentials are also applied at reservoirs 650:652, 654:656, and658:660. This results in a fluid flow along the transverse channels 634,604, 606 and 608, as illustrated by the arrows, and a zero net flowthrough the parallel channel arrays interconnecting these transversechannels, as shown in FIG. 6B. Once the test compound slugs are alignedwith parallel reaction channels 612-620, connecting sample channel 604to detection channel 606, as shown by the shaded areas in FIG. 6B, flowis stopped in all transverse directions by removing the potentialsapplied to the reservoirs at the ends of these channels. As describedabove, the geometry of the channels can be varied to maximize the use ofspace on the substrate. For example, where the sample channel isstraight, the distance between reaction channels (and thus, the numberof parallel reactions that can be carried out in a size limitedsubstrate) is dictated by the distance between sample plugs. Theserestrictions, however, can be eliminated through the inclusion ofaltered channel geometries. For example, in some aspects, the length ofa spacer/guard band plug can be accomodated by a serpentine,square-wave, saw tooth or other reciprocating channel geometry. Thisallows packing a maximum number of reaction channels onto the limitedarea of the substrate surface.

[0095] Once aligned with the parallel reaction channels, the sample isthen moved into the parallel reaction channels 612-620 by applying afirst potential to reservoirs 650 and 652, while applying a secondpotential to reservoirs 658 and 660, whereby fluid flow through parallelpumping channels 636-646 forces the test compounds into parallelreaction channels 612-620, as shown in FIG. 6C. During this process, nopotential is applied at reservoirs 648, 654, 656, or the terminus ofsample channel 604. Parallel channels 636-646 and 622-632 are generallyadjusted in length such that the total channel length, and thus thelevel of resistance, from reservoirs 650 and 652 to channel 604 and fromreservoirs 658 and 660 to channel 606, for any path taken will be thesame. Resistance can generally be adjusted by adjusting channel 35length or width. For example, channels can be lengthened by includingfolding or serpentine geometries. Although not shown as such, toaccomplish this same channel length, channels 636 and 646 would be thelongest and 640 and 642 the shortest, to create symetric flow, therebyforcing the samples into the channels. As can be seen, during flowing ofthe samples through channels 612-620, the resistance within thesechannels will be the same, as the individual channel length is the same.

[0096] Following the reaction to be screened, the sample plug/signalelement is moved into detection channel 606 by applying a potential fromreservoirs 650 and 652 to reservoirs 658 and 660, while the potentialsat the remaining reservoirs are allowed to float. The sampleplugs/signal are then serially moved past the detection window/detector662 by applying potentials to reservoirs 654 and 656, while applyingappropriate potentials at the termini of the other transverse channelsto prevent any flow along the various parallel channels.

[0097] Although generally described in terms of screening assays foridentification of compounds which affect a particular interaction, basedupon the present disclosure, it will be readily appreciated that theabove described microlaboratory systems may also be used to screen forcompounds which specifically interact with a component of a biochemicalsystem without necessarily affecting an interaction between thatcomponent and another element of the biochemical system. Such compoundstypically include binding compounds which may generally be used in,e.g., diagnostic and therapeutic applications as targeting groups fortherapeutics or marker groups, i.e. radionuclides, dyes and the like.For example, these systems may be used to screen test compounds for theability to bind to a given component of a biochemical system.

[0098] III. Microlaboratory System

[0099] Although generally described in terms of individual discretedevices, for ease of operation, the systems described will typically bea part of a larger system which can monitor and control the functioningof the devices, either on an individual basis, or in parallel,multi-device screens. An example of such a system is shown in FIG. 7.

[0100] As shown in FIG. 7, the system may include a test compoundprocessing system 700. The system shown includes a platform 702 whichcan hold a number of separate assay chips or devices 704. As shown, eachchip includes a number of discrete assay channels 706, each having aseparate interface 708, e.g., pipettor, for introducing test compoundsinto the device. These interfaces are used to sip test compounds intothe device, separated by sipping spacer fluid and guard band fluids,into the device. In the system shown, the interfaces of the chip areinserted through an opening 710 in the bottom of the platform 702, whichis capable of being raised and lowered to place the interfaces incontact with test compounds or wash/spacer/guard band fluids, which arecontained in, e.g., multiwell micro plates 711, positioned below theplatform, e.g., on a conveyor system 712. In operation, multiwell platescontaining large numbers of different test compounds are stacked 714 atone end of the conveyor system. The plates are placed upon the conveyorseparated by appropriate buffer reservoirs 716 and 718, which may befilled by buffer system 720. The plates are stepped down the conveyorand the test compounds are sampled into the chips, interspersed byappropriate spacer fluids. After loading the test compounds into thechips, the multiwell plates are then collected or stacked 722 at theopposite end of the system. The overall control system includes a numberof individual microlaboratory systems or devices, e.g., as shown in FIG.7. Each device is connected to a computer system which is appropriatelyprogrammed to control fluid flow and direction within the various chips,and to monitor, record and analyze data resulting from the screeningassays that are performed by the various devices. The devices willtypically be connected to the computer through an intermediate adaptermodule which provides an interface between the computer and theindividual devices for implementing operational instructions from thecomputer to the devices, and for reporting data from the devices to thecomputer. For example, the adapter will generally include appropriateconnections to corresponding elements on each device, e.g., electricalleads connected to the reservoir based electrodes that are used forelectroosmotic fluid flow, power inputs and data outputs for detectionsystems, either electrical or fiberoptic, and data relays for othersensor elements incorporated into the devices. The adapter device mayalso provide environmental control over the individual devices wheresuch control is necessary, e.g., maintaining the individual devices atoptimal temperatures for performing the particular screening assays.

[0101] As shown, each device is also equipped with appropriate fluidinterfaces, e.g., micropipettors, for introducing test compounds intothe individual devices. The devices may be readily attached to roboticsystems which allow test compounds to be sampled from a number ofmultiwell plates that are moved along a conveyor system. Interveningspacer fluids can also be introduced via a spacer solution reservoir.

EXAMPLES

[0102] An assay screen is performed to identify inhibitors of anenzymatic reaction. A schematic of the chip to be used is shown in FIG.8. The chip has a reaction channel 5 cm in length which includes a 1 cmincubation zone and a 4 cm reaction zone. The reservoir at the beginningof the sample channel is filled with enzyme solution and the sidereservoir is filled with the fluorogenic substrate. Each of the enzymeand substrate are diluted to provide for a steady state signal in thelinear signal range for the assay system, at the detector. Potentialsare applied at each of the reservoirs (sample source, enzyme, substrateand waste) to achieve an applied field of 200 V/cm. This applied fieldproduces a flow rate of 2 mm/second. During passage of a given samplethrough the chip, there will generally be a diffusive broadening of thesample. For example, in the case of a small molecule sample, e.g., 1 mMbenzoic acid diffusive broadening of approximately 0.38 mm and anelectrophoretic shift of 0.4 mm is seen.

[0103] Test compound plugs in 150 mM NaCl are introduced into the samplechannel separated by guard bands of 150 mM NaCl and spacer plugs of 5 mMborate buffer. Once introduced into the sample channel shown, samplerequires 12 seconds to travel the length of the sample channel and reachthe incubation zone of the reaction channel. This is a result of theflow rate of 2 mm/sec, allowing for 1 second for moving the samplepipettor from the sample to the spacer compounds. Allowing for theseinteruptions, the net flow rate is 0.68 mm/sec. Another 12 seconds isrequired for the enzyme test compound mixture to travel through theincubation zone to the intersection with the substrate channel wheresubstrate is continuously flowing into the reaction zone of the reactionchannel. Each test compound then requires 48 seconds to travel thelength of the reaction zone and past the fluorescence detector. Aschematic of timing for sample/spacer loading is shown in FIG. 9. Thetop panel shows the sample/spacer/guard band distribution within achannel, whereas the lower panel shows the timing required for loadingthe channel. As shown, the schematic includes the loading (sipping) ofhigh salt (HS) guard band (“A”), moving the pipettor to the sample(“B”), sipping the sample (“C”), moving the pipettor to the high saltguard band solution (“D”) sipping the high salt (“E”), moving thepipettcr to the low salt (LS) spacer fluid (“F”), sipping the low saltspacer (“G”) and returning to the high salt guard band (“H”). Theprocess is then repeated for each additional test compound.

[0104] A constant base fluorescent signal is established at the detectorin the absence of test compounds. Upon introduction of the testcompounds, a decrease in fluorescence is seen which, based upon timedelays corresponds to a specific individual test compound. This testcompound is tentatively identified as an inhibitor of the enzyme, andfurther testing is conducted to confirm this and quantitate the efficacyof this inhibitor.

[0105] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be clear to oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention. All publications and patent documents cited inthis application are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication or patentdocument were so individually denoted.

What is claimed is:
 1. A method of screening a plurality of testcompounds for an effect on a biochemical system, comprising: providing asubstrate having at least a first surface, and at least two intersectingchannels fabricated in said first surface, at least one of said at leasttwo intersecting channels having at least one cross-sectional dimensionin a range from 0.1 to 500 μm; flowing a first component of abiochemical system in a first of said at least two intersectingchannels; flowing at least a first test compound from a second channelinto said first channel whereby said first test compound contacts saidfirst component of said biochemical system; and detecting an effect ofsaid at least first test compound on said biochemical system.
 2. Themethod of claim 1, wherein said at least first component of abiochemical system produces a detectable signal representative of afunction of said biochemical system.
 3. The method of claim 1, whereinsaid at least first component further comprises an indicator compoundwhich interacts with said first component to produce a detectable signalrepresentative of a functioning of said biochemical system.
 4. Themethod of claim 1, wherein said first component of a biochemical systemcomprises an enzyme and a substrate for said enzyme, wherein action ofsaid enzyme on said substrate produces a detectable signal.
 5. Themethod of claim 1, wherein said first component of a biochemical systemcomprises a receptor/ligand binding pair, wherein at least one of saidreceptor or ligand has a detectable signal associated therewith.
 6. Themethod of claim 1, wherein said first component of a biochemical systemcomprises a receptor/ligand binding pair, wherein binding of saidreceptor to said ligand produces a detectable signal.
 7. The method ofclaim 1, wherein said at least first component of a biochemical systemis a biological barrier and said effect of said at least first testcompound is an ability of said test compound to traverse said barrier.8. The method of claim 7, wherein said barrier is selected from thegroup consisting of an epithelial or an endothelial layer.
 9. The methodof claim 1, wherein said at least first component of a biochemicalsystem comprises cells, and said detecting step comprises determining aneffect of said test compound on said cells.
 10. The method of claim 9,wherein said cells are capable of producing a detectable signalcorresponding to a cellular function, and said detecting step comprisesdetecting an effect of said test compound on said cellular function bydetecting a level of said detectable signal.
 11. The method of claim 9,wherein said detecting step comprises detecting an effect of said testcompound on viability of said cells.
 12. A method of screening aplurality of test compounds for an effect on a biochemical system,comprising: providing a substrate having at least a first surface, andat least two intersecting channels fabricated in said first surface, atleast one of said at least two intersecting channels having at least onecross-sectional dimension in a range from 0.1 to 500 μm; continuouslyflowing a first component of a biochemical system in a first channel ofsaid at least two intersecting channels; periodically introducing adifferent test compound into said first channel from a second channel ofsaid at least two intersecting channels; and detecting an effect of saidtest compound on said at least first component of a biochemical system.13. The method of claim 12, wherein said step of periodicallyintroducing comprises flowing a plurality of different test compoundsinto said first channel from a second channel of said at least twointersecting channels, each of said plurality of different testcompounds being physically isolated from each other of said plurality ofdifferent test compounds.
 14. The method of claim 12, wherein said atleast first component of a biochemical system produces a detectablesignal representative of a function of said biochemical system.
 15. Themethod of claim 14, wherein said detecting comprises monitoring saiddetectable signal from said continuously flowing first component at apoint on said first channel, said detectable signal having a steadystate intensity, and wherein said effect of said interaction betweensaid first component and said test compound comprises a deviation fromsaid steady state intensity of said detectable signal.
 16. The method ofclaim 14, wherein said at least first component further comprises anindicator compound which interacts with said first component to producea detectable signal representative of a functioning of said biochemicalsystem.
 17. The method of claim 16, wherein said first component of abiochemical system comprises an enzyme and said indicator compoundcomprises a substrate for said enzyme, wherein action of said enzyme onsaid substrate produces a detectable signal.
 18. The method of claim 14,wherein said at least first component of a biochemical system comprisesa receptor/ligand binding pair, wherein at least one of said receptor orligand has a detectable signal associated therewith.
 19. The method ofclaim 18, wherein said receptor and said ligand flow along said firstchannel at different rates.
 20. The method of claim 14, wherein saidfirst component of a biochemical system comprises a receptor/ligandbinding pair, wherein binding of said receptor to said ligand produces adetectable signal.
 21. The method of claim 12, wherein said at leastfirst component of a biochemical system comprises cells, and saiddetecting step comprises determining an effect of said test compound onsaid cells.
 22. The method of claim 21, wherein said cells are capableof producing a detectable signal corresponding to a cellular function,and said detecting step comprises detecting an effect of said testcompound on said cellular function by detecting a level of saiddetectable signal.
 23. The method of claim 21, wherein said detectingstep comprises detecting an effect of said test compound on viability ofsaid cells.
 24. A method of screening a plurality of different testcompounds for an effect on a biochemical system, comprising: providing asubstrate having at least a first surface, and a plurality of reactionchannels fabricated in said first surface, each of said plurality ofreaction channels being fluidly connected to at least two transversechannels fabricated in said surface; introducing at least a firstcomponent of a biochemical system into said plurality of reactionchannels; flowing a plurality of different test compounds through atleast one of said at least two transverse channels, each of saidplurality of test compounds being introduced into said at least onetransverse channels in a discrete volume; directing each of saidplurality of different test compounds into a separate one of saidplurality of reaction channels; and detecting an effect of each of saidtest compounds on said at least one component of said biochemicalsystem.
 25. The method of claim 24, wherein said at least firstcomponent of said biochemical system produces a flowable detectablesignal representative of a function of said biochemical system.
 26. Themethod of claim 25, wherein said detectable flowable signal produced ineach of said plurality of reaction channels is flowed into and throughsaid second transverse channel, each of said detectable flowable signalsproduced in each of said plurality of reaction channels being physicallyisolated from each other of said detectable flowable signals, whereuponeach of said detectable flowable signals is separately detected.
 27. Themethod of claim 25, wherein said flowable signal comprises a solublesignal.
 28. The method of claim 27, wherein said soluble signal isselected from fluorescent or colorimetric signals.
 29. The method ofclaim 24, wherein said at least first component further comprises anindicator compound which interacts with said first component to producea detectable signal representative of a functioning of said biochemicalsystem.
 30. The method of claim 29, wherein said first component of abiochemical system comprises an enzyme and said indicator compoundcomprises a substrate for said enzyme, wherein action of said enzyme onsaid substrate produces a detectable signal.
 31. The method of claim 24,wherein said at least first component of a biochemical system comprisesa receptor/ligand binding pair, wherein at least one of said receptor orligand has a detectable signal associated therewith.
 32. The method ofclaim 24, wherein said first component of a biochemical system comprisesa receptor/ligand binding pair, wherein binding of said receptor to saidligand produces a detectable signal.
 33. The method of claim 24, whereinsaid at least first component of a biochemical system comprises cells,and said detecting step comprises determining an effect of said testcompound on said cells.
 34. The method of claim 33, wherein said cellsare capable of producing a detectable signal corresponding to a cellularfunction, and said detecting step comprises detecting an effect of saidtest compound on said cellular function by detecting a level of saiddetectable signal.
 35. The method of claim 34, wherein said detectingstep comprises detecting an effect of said test compound on viability ofsaid cells.
 36. The method of claim 24, wherein each of said pluralityof different test compounds is immobilized upon a separate bead, andsaid step of directing each of said plurality of different testcompounds into a separate one of said plurality of reaction channelscomprises: lodging one of said separate beads at an intersection of saidfirst transverse channel and each of said plurality of reactionchannels; and controllably releasing said test compounds from each ofsaid separate beads into each of said plurality of reaction channels.37. An apparatus for screening test compounds for an effect on abiochemical system, comprising: a substrate having at least one surface;at least two intersecting channels fabricated into said surface of saidsubstrate, at least one of said at least two intersecting channelshaving at least one cross-sectional dimension in the range from about0.1 to about 500 μm; a source of a plurality different test compoundsfluidly connected to a first of said at least two intersecting channels;a source of at least one component of said biochemical system fluidlyconnected to a second of said at least two intersecting channels; afluid direction system for flowing said at least one component withinsaid second of said at least two intersecting channels and forintroducing said different test compounds from said first to said secondof said at least two intersecting channels; a cover mated with saidsurface; and a detection zone in said second channel for detecting aneffect of said test compound on said biochemical system.
 38. Theapparatus of claim 37, wherein said fluid direction system generates acontinuous flow of said at least first component along said second ofsaid at least two intersecting channels, and periodically injects a testcompound from said first channel into said second channel.
 39. Theapparatus of claim 37, further comprising a source of a second componentof said biochemical system, and a third channel fabricated into saidsurface, said third channel fluidly connecting at least one of said atleast two intersecting channels with said source of said secondcomponent of said biochemical system.
 40. The apparatus of claim 39,wherein said fluid direction system generates a continuous flow of amixture of said first component and said second component along saidsecond of said at least two intersecting channels, and periodicallyinjects a test compound from said first channel into said secondchannel.
 41. The apparatus of claim 37, wherein said fluid directionsystem continuously flows said plurality of different test compoundsfrom said first into said second of said at least two intersectingchannels, each of said plurality of different test compounds beingseparated by a fluid spacer.
 42. The apparatus of claim 37, wherein saidfluid direction system comprises: at least three electrodes, eachelectrode being in electrical contact with said at least twointersecting channels on a different side of an intersection formed bysaid at least two intersecting channels; and a control system forconcomitantly applying a variable voltage at each of said electrodes,whereby movement of said test compounds or said at least first componentin said at least two intersecting channels may be controlled.
 43. Theapparatus of claim 37, wherein said detection system includes adetection window in said second channel.
 44. The apparatus of claim 43,wherein said detection system is a fluorescent detection system.
 45. Theapparatus of claim 37, wherein said substrate is planar.
 46. Theapparatus of claim 37, wherein said substrate comprises etched glass.47. The apparatus of claim 37, wherein said substrate comprises etchedsilicon.
 48. The apparatus of claim 37, further comprising an insulatinglayer disposed over said etched silicon substrate.
 49. The apparatus ofclaim 37, wherein said substrate is a molded polymer.
 50. The apparatusof claim 37, wherein said at least one component of a biochemical systemcomprises an enzyme, and a substrate which produces a detectable signalwhen reacted with said enzyme.
 51. The apparatus of claim 50, whereinsaid substrate is selected from the group consisting of chromogenic andfluorogenic substrates.
 52. The apparatus of claim 37, wherein said atleast first component of a biochemical system comprises areceptor/ligand binding pair, wherein at least one of said receptor orligand has a detectable signal associated therewith.
 53. The apparatusof claim 37, wherein said first component of a biochemical systemcomprises a receptor/ligand binding pair, wherein binding of saidreceptor to said ligand produces a detectable signal.
 54. An apparatusfor detecting an effect of a test compound on a biochemical system,comprising: a substrate having at least one surface; a plurality ofreaction channels fabricated into said surface; at least two transversechannels fabricated into said surface, each of said plurality ofreaction channels being fluidly connected to a first of said at leasttwo transverse channels at a first point in said reaction channels, andfluidly connected to a second of said at least two transverse channelsat a second point in said reaction channels, said at least twotransverse channels and said plurality of reaction channels each havingat least one cross-sectional dimension in the range from about 0.1 toabout 500 μm; a source of at least one component of said biochemicalsystem, said source of at least one component of said biochemical systembeing fluidly connected to each of said plurality of reaction channels;a source of test compounds fluidly connected to said first of said atleast two transverse channels; a fluid direction system for controllingmovement of said test compound and said at least one component withinsaid at least two transverse channels and said plurality of reactionchannels; a cover mated with said surface; and a detection system fordetecting an effect of said test compound on said biochemical system.55. The apparatus of claim 54, wherein said fluid control systemcomprises: a plurality of individual electrodes, each in electricalcontact with each terminus of said at least two transverse channels; anda control system for concomitantly applying a variable voltage at eachof said electrodes, whereby movement of said test compounds or said atleast first component in said at least two transverse channels and saidplurality of reaction channels may be controlled.
 56. The apparatus ofclaim 54, wherein each of said plurality of reaction channels comprisesa bead resting well at said first point in said plurality of reactionchannels.
 57. The apparatus of claim 54, wherein said source of at leastone component of a biochemical system is fluidly connected to saidplurality of reaction channels by a third transverse channel, said thirdtransverse channel having at least one cross sectional dimension in arange of from 0.1 to 500 μm and being fluidly connected to each of saidplurality of reaction channels at a third point in said reactionchannels.
 58. The apparatus of claim 57, wherein said third point insaid reaction channels is intermediate to said first and second pointsin said reaction channels.
 59. The apparatus of claim 58, furthercomprising a particle retention zone in each of said plurality ofreaction channels, between said third and said second points in saidplurality of reaction channels.
 60. The apparatus of claim 49, whereinsaid particle retention zone comprises a particle retention matrix. 61.The apparatus of claim 49, wherein said particle retention zonecomprises a microstructural filter.
 62. The apparatus of claim 54,wherein said plurality of reaction channels comprises a plurality ofparallel reaction channels fabricated into said surface of saidsubstrate and said at least two transverse channels are connected atopposite ends of each of said parallel reaction channels.
 63. Theapparatus of claim 54, wherein said at least two transverse channels arefabricated on said surface of said substrate in inner and outerconcentric channels, and said plurality of reaction channels extendradially from said inner concentric channel to said outer concentricchannel.
 64. The apparatus of claim 63, wherein said detection systemcomprises a detection window in said second channel.
 65. The apparatusof claim 64, wherein said detection system is a fluorescent detectionsystem.
 66. The apparatus of claim 54, wherein said substrate is planar.67. The apparatus of claim 54, wherein said substrate comprises etchedglass.
 68. The apparatus of claim 54, wherein said substrate comprisesetched silicon.
 69. The apparatus of claim 54, further comprising aninsulating layer disposed over said etched silicon substrate.
 70. Theapparatus of claim 54, wherein said substrate is a molded polymer. 71.The apparatus of claim 54, wherein said at least one component of abiochemical system comprises an enzyme, and an enzyme substrate whichproduces a detectable signal when reacted with said enzyme.
 72. Theapparatus of claim 71, wherein said enzyme substrate is selected fromthe group consisting of chromogenic and fluorogenic substrates.
 73. Theapparatus of claim 54, wherein said at least first component of abiochemical system comprises a receptor/ligand binding pair, wherein atleast one of said receptor or ligand has a detectable signal associatedtherewith.
 74. The apparatus of claim 54, wherein said first componentof a biochemical system comprises a receptor/ligand binding pair,wherein binding of said receptor to said ligand produces a detectablesignal.